摘要
目的探讨神经细胞黏附分子(neural cell adhesion molecule,NCAM)衍生肽P2对缺血性脑卒中大鼠神经功能损伤的修复作用及机制。方法提取并培养原代皮质神经元,氧糖剥夺(oxygen-glucose deprivation,OGD)造模后采用CCK-8法观察不同浓度P2对OGD条件下皮质神经元活性的影响,Western blot检测凋亡相关蛋白及细胞外信号调节激酶1/2(extracellular signal regulated kinase 1/2,Erk1/2)的表达。选取清洁级雄性SD大鼠进行动物实验,采用大脑中动脉线栓法(middle cerebral artery occlusion,MCAO)建立大鼠脑卒中缺血/再灌注损伤动物模型,造模成功的大鼠按照随机数字表法分为假手术组、MCAO组和MCAO+P2组,每组12只。术后MCAO+P2组大鼠每日一次皮下注射1 mg/kg P2,持续至术后14 d,其余两组大鼠均皮下注射等体积0.9%氯化钠溶液。平衡木实验观察大鼠运动功能损伤及恢复情况。免疫荧光染色检测大鼠脑梗死灶周边的神经元凋亡比率,Western blot检测大鼠脑梗死灶周边脑组织内Erk1/2蛋白的表达。采用SPSS 22.0进行统计分析,平衡木实验数据采用重复测量方差分析,其他实验数据以单因素方差分析进行检验。结果与OGD组相比,0.5,1.0和2.0μmol/L的P2均可以提高OGD条件下神经元细胞活性,其中1μmol/L的P2效果最好(2.436±0.284,1.551±0.410,P<0.05)。Western blot结果显示,加入1μmol/L P2后凋亡相关蛋白bax[(76.120±3.232)%,(88.965±5.208)%,P<0.05]、cleaved caspase-3[(76.736±4.306)%,(97.781±8.111)%,P<0.05]和cleaved caspase-9[(88.833±6.581)%,(104.962±4.788)%,P<0.05]表达均低于OGD组,bcl-2[(56.146±3.882)%,(43.170±6.945)%,P<0.05]以及磷酸化Erk1/2蛋白[(73.583±8.557)%,(55.219±4.615)%,P<0.05]表达均高于OGD组。与MCAO组相比,P2干预后第14天时大鼠瘫痪侧后肢的滑脱率低[(23.438±11.540)%,(41.733±13.631)%,P<0.05],病灶周边神经元凋亡比率低[(13.144±6.485)%,(26.699±6.402)%,P<0.05],大鼠梗死灶周边脑组织内磷酸化Erk1/2蛋白�
Objective To clarify the neuroprotective effects of neural cell adhesion molecule(NCAM)derived peptide P2 on in vitro cultured neuron and ischemic stroke rat.Methods Primary cortical neurons were extracted and cultured,and CCK-8 method was used to observe the protective effect of different concentrations of P2 on cortical neurons under oxygen-glucose deprivation(OGD)conditions.The levels of apoptosis-related proteins and extracellular signal regulated kinase 1/2(Erk1/2)were observed by Western blot.Clean grade male SD rats were selected for animal experiments.The middle cerebral artery occlusion(MCAO)method was used to establish the rat model of cerebral ischemia/reperfusion injury.The rats with successful model were divided into sham operation group,MCAO group and MCAO+P2 group according to the random number table,with 12 rats in each group.After operation,rats in MCAO+P2 group were subcutaneously injected with 1 mg/kg P2 once a day until 14 days after operation,and rats in the other two groups were subcutaneously injected with 0.9%sodium chloride solution of the same volume.Beam-walking test was used to evaluate the motor function of rats.Immunofluorescence staining and Western blot were used to detect the in-situ apoptosis of neuronal cells and the expression of Erk1/2 in ischemic penumbra of rat brains,respectively.All statistical analyses were performed using SPSS 22.0.Repeated measurement ANOVA was used to evaluate the beam-walking experimental data,and one-way ANOVA were used to analyze other experimental data among multiple groups.Results Compared with OGD group,0.5,1.0 and 2.0μmol/L P2 improved the activity of neurons under OGD conditions,of which 1μmol/L P2 had the best effect((2.436±0.284),(1.551±0.410),P<0.05).Western blot showed that the protein levels of bax((76.120±3.232)%,(88.965±5.208)%,P<0.05),cleaved caspase-3((76.736±4.306)%,(97.781±8.111)%,P<0.05)and cleaved caspase-9((88.833±6.581)%,(104.962±4.788)%,P<0.05)in 1μmol/L P2 treated group were all lower than those in OGD group,while th
作者
蓝晓艳
孙正武
储成艳
赵梦微
李深
Lan Xiaoyan;Sun Zhengwu;Chu Chengyan;Zhao Mengwei;Li Shen(Department of Neurointervention and Intensive Care Unit,Dalian Municipal Central Hospital,Dalian 116033,China;Department of Clinical Pharmacy,Dalian Municipal Central Hospital,Dalian 116033,China;Department of Neurology,Dalian Municipal Central Hospital,Dalian 116033,China;Department of Neurology and Psychiatry,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2022年第11期968-975,共8页
Chinese Journal of Behavioral Medicine and Brain Science
基金
大连市医学科学研究计划项目(1811014)。
关键词
神经细胞黏附分子衍生肽P2
缺血性脑卒中
氧糖剥夺
细胞外信号调节激酶
凋亡
大鼠
Neural cell adhesion molecule derived peptide P2
Ischemic stroke
Oxygen-glucose deprivation
Extracellular signal regulated kinase 1/2
Apoptosis
Rats
